Illumina dye sequencing
Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris. It was developed by Shankar Balasubramanian and David Klenerman of Cambridge University, who subsequently founded Solexa, a company later acquired by Illumina. This sequencing method is based on reversible dye-terminators that enable the identification of single bases as they are introduced into DNA strands. It can also be used for whole-genome and region sequencing, transcriptome analysis, metagenomics, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis.
Illumina sequencing technology works in three basic steps: amplify, sequence, and analyze. The process begins with purified DNA. The DNA gets chopped up into smaller pieces and given adapters, indices, and other kinds of molecular modifications that act as reference points during amplification, sequencing, and analysis. The modified DNA is loaded onto a specialized chip where amplification and sequencing will take place. Along the bottom of the chip are hundreds of thousands of oligonucleotides (short, synthetic pieces of DNA). They are anchored to the chip and able to grab DNA fragments that have complementary sequences. Once the fragments have attached, a phase called cluster generation begins. This step makes about a thousand copies of each fragment of DNA. Next, primers and modified nucleotides enter the chip. These nucleotides have reversible 3' blockers that force the polymerase to add on only one nucleotide at a time as well as fluorescent tags. After each round of synthesis, a camera takes a picture of the chip. A computer determines what base was added by the wavelength of the fluorescent tag and records it for every spot on the chip. After each round, non-incorporated molecules are washed away. A chemical deblocking step is then used in the removal of the 3’ terminal blocking group and the dye in a single step. The process continues until the full DNA molecule is sequenced. With this technology, thousands of places throughout the genome are sequenced at once via massive parallel sequencing.
The first step after DNA purification is tagmentation. Transposases randomly cut the DNA into short segments ("tags"). Adapters are added on either side of the cut points (ligation). Strands that fail to have adapters ligated are washed away.
Reduced cycle amplification
The next step is called reduced cycle amplification. During this step, sequences for primer binding, indices, and terminal sequences are added. Indices are usually six base pairs long and are used during DNA sequence analysis to identify samples. Indices allow for up to 96 different samples to be run together. During analysis, the computer will group all reads with the same index together. The terminal sequences are used for attaching the DNA strand to the flow cell. Illumina uses a "sequence by synthesis" approach. This process takes place inside of an acrylamide-coated glass flow cell. The flow cell has oligonucleotides (short nucleotide sequences) coating the bottom of the cell, and they serve to hold the DNA strands in place during sequencing. The oligos match the two kinds of terminal sequences added to the DNA during reduced cycle amplification. As the DNA enters the flow cell, one of the adapters attaches to a complementary oligo.
Once attached, cluster generation can begin. The goal is to create hundreds of identical strands of DNA. Some will be the forward strand; the rest, the reverse. Clusters are generated through bridge amplification. Polymerases move along a strand of DNA, creating its complementary strand. The original strand is washed away, leaving only the reverse strand. At the top of the reverse strand there is an adapter sequence. The DNA strand bends and attaches to the oligo that is complementary to the top adapter sequence. Polymerases attach to the reverse strand, and its complementary strand (which is identical to the original) is made. The now double stranded DNA is denatured so that each strand can separately attach to an oligonucleotide sequence anchored to the flow cell. One will be the reverse strand; the other, the forward. This process is called bridge amplification, and it happens for thousands of clusters all over the flow cell at once.
Over and over again, DNA strands will bend and attach to oligos. Polymerases will synthesize a new strand to create a double stranded segment, and that will be denatured so that all of the DNA strands in one area are from a single source (clonal amplification). Clonal amplification is important for quality control purposes. If a strand is found to have an odd sequence, then scientists can check the reverse strand to make sure that it has the complement of the same oddity. The forward and reverse strands act as checks to guard against artifacts. Because Illumina sequencing uses polymerases, base substitution errors have been observed, especially at the 3' end. Paired end reads combined with cluster generation can confirm an error took place. The reverse and forward strands should be complementary to each other, all reverse reads should match each other, and all forward reads should match each other. If a read is not similar enough to its counterparts (with which it should be a clone), an error may have occurred. A minimum threshold of 97% similarity has been used in some labs' analyses.
Sequence by synthesis
At the end of clonal amplification, all of the reverse strands are washed off the flow cell, leaving only forward strands. Primers attach to the forward strands and a polymerase adds fluorescently tagged nucleotides to the DNA strand. Only one base is added per round. A reversible terminator is on every nucleotide to prevent multiple additions in one round. Using the four-colour chemistry, each of the four bases has a unique emission, and after each round, the machine records which base was added. Starting with the launch of the NextSeq and later the MiniSeq, Illumina introduced a new two-colour sequencing chemistry. Nucleotides are distinguished by either one of two colours (red or green), no colour ("black") or binding both colours (appearing orange as a mixture between red and green).
Once the DNA strand has been read, the strand that was just added is washed away. Then, the index 1 primer attaches, polymerizes the index 1 sequence, and is washed away. The strand forms a bridge again, and the 3' end of the DNA strand attaches to an oligo on the flow cell. The index 2 primer attaches, polymerizes the sequence, and is washed away.
A polymerase sequences the complementary strand on top of the arched strand. They separate, and the 3' end of each strand is blocked. The forward strand is washed away, and the process of sequence by synthesis repeats for the reverse strand.
The sequencing occurs for millions of clusters at once, and each cluster has ~1,000 identical copies of a DNA insert. The sequence data is analyzed by finding fragments with overlapping areas, called contigs, and lining them up. If a reference sequence is known, the contigs are then compared to it for variant identification.
This piecemeal process allows scientists to see the complete sequence even though an unfragmented sequence was never run; however, because Illumina read lengths are not very long (HiSeq sequencing can produce read lengths around 90 bp long), it can be a struggle to resolve short tandem repeat areas. Also, if the sequence is de novo and so a reference doesn't exist, repeated areas can cause a lot of difficulty in sequence assembly. Additional difficulties include base substitutions (especially at the 3' end of reads) by inaccurate polymerases, chimeric sequences, and PCR-bias, all of which can contribute to generating an incorrect sequence.
Comparison with other sequencing methods
This technique offers a number of advantages over traditional sequencing methods such as Sanger sequencing. Due to the automated nature of Illumina dye sequencing it is possible to sequence multiple strands at once and gain actual sequencing data quickly. Additionally, this method only uses DNA polymerase as opposed to multiple, expensive enzymes required by other sequencing techniques (i.e. pyrosequencing).
Examples of use
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